EP3950721A1 - Pullulan-containing powder - Google Patents

Pullulan-containing powder Download PDF

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Publication number
EP3950721A1
EP3950721A1 EP21192842.9A EP21192842A EP3950721A1 EP 3950721 A1 EP3950721 A1 EP 3950721A1 EP 21192842 A EP21192842 A EP 21192842A EP 3950721 A1 EP3950721 A1 EP 3950721A1
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EP
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Prior art keywords
pullulan
particulate composition
weight
content
saccharides
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Pending
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EP21192842.9A
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German (de)
English (en)
French (fr)
Inventor
Takashi Shibuya
Seisuke Izawa
Toshio Miyake
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Nagase Viita Co Ltd
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Hayashibara Co Ltd
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Publication of EP3950721A1 publication Critical patent/EP3950721A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C12P19/10—Pullulan
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
    • C08B37/0018—Pullulan, i.e. (alpha-1,4)(alpha-1,6)-D-glucan; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/0216—Solid or semisolid forms
    • A61K8/022—Powders; Compacted Powders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/732—Starch; Amylose; Amylopectin; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/02—Dextran; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to a particulate composition containing pullulan, process for producing the same, and uses thereof; and more particularly, to a novel particulate composition containing pullulan, which can be produced at a lesser cost without employing any complicated purification step and has advantageous features, process for producing the same, and uses thereof.
  • pullulan is a colorless and odorless water-soluble polysaccharide, a linear ⁇ -glucan basically consisting of repeating units of maltotriose linked together in an ⁇ -1, 6 linkage fashion, produced by a microorganism of the species Aureobasidium pullulans , a kind of fungus.
  • Pullulan is a safe and edible polysaccharide produced from the microorganism usually by assimilating saccharides such as starch or starch syrup as carbon sources.
  • pullulan Since pullulan has properties such as satisfactory water-solubility, relatively high adhesiveness, and improved film-formability, it has been extensively used in the fields of cosmetics and pharmaceuticals, as well as food products, in such a manner of using as an adhesive or coating material for food products and pharmaceuticals, a shaped products such as edible films and sheets dispersed with ingredients such as dyes, and as capsules encapsulated with granules containing functional ingredients used in the fields of food products and pharmaceuticals.
  • Japanese Patent Publication No. 4291/84 and Japanese Patent Laid-OpenNos. 111817/84 and 1993-285969 disclose processes for producing pullulan films;
  • Japanese Patent No. 3350823 discloses an adhesive composed of pullulan as a main ingredient;
  • Japanese Patent Publication No. 6346/77 discloses a method for preventing oxidation using a pullulan film or pullulan coating;
  • 16217/85 discloses a film sweetener for diet prepared by dispersing a sweetener into a pullulan film; and Japanese Patent Publication No. 553/95 and Japanese Patent Laid-Open No. 2005-21124 disclose film preparations prepared by dispersing agents into pullulan; Satoshi NAKAMURA, "Yu-Kagaku” (Oil Chemicals), Vol. 34, No. 10, pp. 865-871, 1985 , discloses an overview on the method for preparation of a pullulan membrane; and Satoshi NAKAMURA, "Japan Food Science", Vol. 25, No. 4, pp. 61-67, 1986 , " Food Industry", Vol. 30, No. 10, pp.
  • Pullulan is formed in the culture of the above-identified microorganism, and the resulting culture containing pullulan is purified through the steps of removing the cells of the microorganims by centrifugation or filtration, and if necessary, after decoloring and desalting the resultant, adding thereunto an organic solvent such as methanol. Since pullulan is insoluble in methanol and concomitant saccharides other than pullulan are usually soluble in aqueous methanol, an organic solvent such as methanol is added to the above-identified culture to precipitate pullulan to separate into precipitant and supernatant. Thus, concomitant saccharides can be removed.
  • pullulan is purified and improved its purity.
  • the purified pullulan is prepared into a particulate composition containing pullulan via an appropriate pulverization step.
  • Japanese Patent Laid-Open No. 21739/83 and Japanese Patent Nos. 27099/80 and 50665/83 disclose processes for producing shaped products such as films, coating membranes, sheets, and capsules which use the pullulan that has been purified and produced as in the above.
  • a relatively large amount of organic solvent is required in the purification step for pullulan by solvent precipitation where an organic solvent is added to a culture containing pullulan to precipitate pullulan which is then separated from the supernatant. Therefore, even if the used organic solvent is recovered by means of distillation, etc., the cost for apparatuses and organic solvents required for such recovering is not negligible, resulting in an unsatisfactory increment of its production cost.
  • the purification step in itself for precipitating and separating pullulan is laborious and it requires time and effort, and after all this results in an increment of price of the resulting particulate composition containing pullulan.
  • the particulate composition containing pullulan produced by the above purification step is, for example, as disclosed by Satoshi NAKAMURA in "Yu-Kagaku” (Oil Chemicals), Vol. 34, No. 10, pp. 865-871, 1985 , " Japan Food Science", Vol. 25, No. 4, pp. 61-67, 1986 , " Food Industry", Vol. 30, No. 10, pp. 33-39, 1987, Korin Publishing Co., Ltd., Tokyo, Japan , and Japanese Patent Publication No. 49164/85 , limited to specific uses for "reagent-grade pullulan” or "standard sample of pullulan” for molecular standard for assaying molecular weight, and "plasma adjuvant" in the field of pharmaceuticals.
  • Japanese Patent No. 3012917 discloses a pullulan separated and purified on high-performance liquid chromatography in normal-separation mode using a column packed with an amide-linked silica.
  • Japanese Patent No. 3232488 a process for producing a high pullulan content product with an increased pullulan content of up to at least about 50% by weight, on a dry solid basis (d.s.b.), preferably, about 55% to about 90% by weight. Since the product can be produced without employing any complicated purification step by solvent sedimentation, it has an advantageous feature that it can be produced easily at a lesser cost.
  • Japanese Patent No. 4203322 the same applicant as the present invention discloses a high pullulan content product with an improved stability against moisture change by adding thereunto ⁇ , ⁇ -trehalose; in Japanese Patent Laid-Open No.
  • Food Additive Pullulan Since the “Food Additive Pullulan” has a satisfactory adhesive power, adherence, membrane-formability, and lubricity, as well as safeness and relatively low price, it has been extensively used as general-purpose particulate compositions containing pullulan; food viscosity-imparting agents, bonds, adhesives, and coating agents; instantly water-soluble solid-color films after processing; print films being printed with edible inks; and as blended films kneaded with food products, flavors, etc.
  • pullulan films prepared with the above-identified "Food Additive Pullulan” have various improved properties but have the following defects of causing occasional cracking or rupturing to become unusable when a stress concentrated site were formed by folding, inflecting or the like.
  • the "Food Additive Pullulan” reaches its pullulan content of at least 90% by weight, the content or the molecular distribution of pullulan contained therein is not constant depending on its production lot, and thus it has the defect that products with constant properties of solubility and adhesive power could hardly be obtained.
  • the pullulan content in "Food Additive Pullulan” is at least 90% by weight; however, it changes usually in the range of about 90% to about 96% by weight, depending on its production lot. Following this change, the content of concomitant saccharides other than pullulan will change in the range of about 4% to about 10% by weight.
  • the fluctuation range of the content of concomitant saccharides changes even by two times or higher, it naturally influences on the solubility and adhesiveness of a particulate composition containing pullulan, resulting in the defect that a desired product with constant solubility, adhesive power, or the like could hardly be obtained.
  • pharmaceutical additives used for such pharmaceuticals should be those which do not affect pharmacological effects but stably maintain their dispositions.
  • "Food Additive Pullulan” when conventional "Food Additive Pullulan" is used as a pharmaceutical additive, for example, as an adhesive agent for tablets, etc., as pharmaceuticals, the dissolution rate of the effective ingredients of the pharmaceuticals could not be constant depending on its production lot, and this has a fear of affecting pharmacokinetics in living bodies.
  • Food Additive Pullulan should not necessarily be sufficient enough for an additive for use as pharmaceuticals which require persistent properties such as strength, solubility, and adhesive power and also require a constant disposition; or for use as quasi-drugs and cosmetics.
  • the present invention which was made to solve the above defect residing in conventional particulate composition containing pullulan, aims to provide a particulate composition containing pullulan that is produced without employing any complicated purification step by solvent sedimentation or the like, has a higher rupture strength than those of conventional ones when formed into a film, has a constant composition retaining stably its concomitant saccharide content to an extremely low level, and preferably, has a molecular distribution of pullulan contained therein; and to provide uses thereof.
  • the present inventors energetically studied to solve the above object and repeatedly took a process of trial and error to find specific mutants of a microorganism of the species Aureobasidium pullulans, which yield pullulan in an amount substantially equal to that of the parent strain but have a significantly lesser ratio of saccharides other than pullulan, i.e., concomitant saccharides, among the artificially produced microorganisms of the species Aureobasidium pullulans when cultured in a selection medium containing glucose and maltose as carbon sources.
  • the one with the minimum ratio of concomitant saccharides was selected, cultured in a culture medium containing glucose and maltose as carbon sources, followed by producing a particulate composition containing pullulan in usual manner without employing any purification step of solvent precipitation for precipitating pullulan with an organic solvent.
  • a particulate composition containing pullulan with a significantly lesser amount of mannitol, ametabolite of a microorganism of the species Aureobasidium pullulans compared to the above-identified "Food Additive Pullulan”.
  • the rupture strength was significantly higher than that obtained with the above "FoodAdditive Pullulan".
  • the concomitant saccharide content in the particulate composition remains stably at an extremely low level independently of its production lot, the saccharide composition remains roughly stable, and the molecular distribution of pullulan contained in the composition remains stably within a constant range. Therefore, the particulate composition can keep its quality at a constant level in terms of the above rupture strength, as well as solubility against water and adhesive power to bind other substances each other.
  • the particulate composition affords the shaped products a constant quality in terms of physical properties such as strength, solubility, and disintegrability.
  • the particulate composition containing pullulan can form shaped products with constant and persistent physical properties required for pharmaceuticals, quasi-drugs, cosmetics, etc., and particularly, even when used in pharmaceuticals, the particulate composition can be used as a pharmaceutical additive because it has no fear of affecting disposition of effective ingredients that is inducible by dispersion of dissolution or disintegration.
  • the present invention was made based on these findings.
  • the present invention solves the above object by providing a particulate composition containing pullulan characterized in that it is produced by culturing a mutant of a microorganism of the species Aureobasidium pullulans in a culture medium containing glucose and maltose as carbon sources without employing a step of removing concomitant saccharides from the resulting culture medium; it consists of a pullulan fraction insoluble in 75% by volume of methanol in water and a fraction of concomitant saccharides soluble in the aqueous methanol solution; it has a ratio of 3% by weight or lower of concomitant saccharides in terms of the concomitant saccharide content against the total content of sugars contained in the whole particulate composition, determined based on the anthrone-sulfuric acid method; and that it contains mannitol.
  • the particulate composition containing pullulan has a significant feature of that it has a lesser ratio of 3% by weight or lower of concomitant saccharides in terms of the concomitant saccharide content against the total content of sugars contained in the whole particulate composition, and the above total saccharide content and the concomitant saccharide content are both determined based on the anthrone-sulfuric acid method.
  • the anthrone-sulfuric acid method is an assay system for saccharides where anthrone color reaction is utilized, and the absorbance of the color reaction is obtained as an amount proportional to the saccharide content, and therefore the saccharide content can be obtained based on the data.
  • saccharides which consist of glucose they can be determined in terms of D-glucose by using D-glucose as a standard substance.
  • the anthrone-sulfuric acid method is employed as an assay for "Monosaccharides and Oligosaccharides” in purification test for pullulan as disclosed in Japanese Standard of Food Additives" (8th edition), published by Japan Food Additives Association (JAFA), pp. 572-573 (see the column of "Pullulan”), 2007 .
  • saccharides for example, like the later described mannitol, there exist saccharides that are not colored and undetectable by the anthrone-sulfuric acid method, and the total saccharide content and the concomitant saccharide content do not necessarily correspond exactly with the total saccharide content and the concomitant saccharide content.
  • the total saccharide content determined based on the anthrone-sulfuric acid method is simply called “total saccharide content", unless specified otherwise; and also the concomitant saccharide content in the concomitant saccharide fraction, determined based on the method, is simply called “concomitant saccharide content”.
  • the pullulan content in a particulate composition containing pullulan can be determined by assaying the pullulan content in a pullulan fraction based on the anthrone-sulfuric acid method in accordance with the assay for concomitant saccharide content, however, since the particulate composition containing pullulan of the present invention consists of a pullulan fraction and a concomitant saccharide fraction, the pullulan content of the particulate composition can be easily determined by subtracting the above concomitant saccharide content from the above total saccharide content.
  • the ratio of the concomitant saccharide content against the total saccharide content is 3% by weight or lower, and this means that the pullulan content in a pullulan fraction is 97% by weight or higher.
  • the pullulan content does not contain saccharides such as mannitol that are not detected by the anthrone-sulfuric acid method.
  • the particulate composition containing pullulan of the present invention contains concomitant saccharides whose maximum level is extremely as low as 3% by weight of the total saccharide content, if the concomitant saccharide content varies from over 0% by weight to 3% by weight, the changing ratio against the remaining 97% by weight or higher of pullulan is relatively low, and therefore the total saccharide composition of the particulate composition containing pullulan of the present invention is roughly constant as a whole.
  • the particulate composition of the present invention has another maj or feature that it contains mannitol.
  • mannitol is a substance that is contained in the culture as a metabolite of a microorganism of the species Aureobasidium pullulans.
  • mannitol is soluble in aqueous methanol and is removed when the resulting culture is purified by solvent precipitation using an organic solvent(s).
  • the fact that mannitol is not removed from the particulate composition and contained therein means that the particulate composition containing pullulan of the present invention is of that produced without employing a step of removing concomitant saccharides by solvent precipitation using organic solvents, etc.
  • the particulate composition usually contains about 2% by weight, d.s.b., of saccharides that are not colored and detected by the anthrone-sulfuric acid method, and the main ingredient is mannitol.
  • mannitol is quantified, for example, on high-performance liquid chromatography (HPLC) analysis shown in the later described Experiment 4. Since the detection limit of mannitol on the HPLC analysis is usually speculated to be about 0.01% by weight, the fact that the particulate composition containing pullulan of the present invention means that it contains at least 0.01% by weight, d.s.b., of mannitol. Depending on the variety of the types of carbon sources used in culture, the content of mannitol in the particulate composition containing pullulan of the present invention does not usually exceed 2% by weight, d.s.b.
  • HPLC high-performance liquid chromatography
  • Mannitol dissolves in 75% by volume of methanol in water and usually it is contained in a concomitant saccharide fraction, however, as mentioned above, mannitol is not contained in the content of concomitant saccharides because it is not detected on the anthrone-sulfuric acid method.
  • the particulate composition containing pullulan of the present invention contains mannitol and is produced without employing a step of removing concomitant saccharides by solvent precipitation using an organic solvent(s), etc., the concomitant saccharide content is extremely as low as 3% by weight or lower. Accordingly, the particulate composition, which contains mannitol and has an extremely lower amount of concomitant saccharides, has not yet been provided before the present invention was made as far as the applicant of the present invention knows and thus it is a novel particulate composition containing pullulan.
  • the particulate composition containing pullulan of the present invention can be formed into a film with a thickness of 100 ⁇ m, more particularly, a film with a thickness of 100 ⁇ m is obtained by casting a 20% w/v aqueous solution of the particulate composition on a plain plate, drying the casted solution at 25°C, and allowing the dried product to stand at a relative humidity of 22%.
  • the film thus obtained exhibits a rupture strength of piercing of 20 MPa or higher on the piercing test for rupture strength using an adaptor for piercing test having a sectional area of 1 mm 2 .
  • the above-identified rupture strength of piercing is significantly higher than that obtained with the above-identified "Food Additive Pullulan".
  • the fact shows that the particulate composition containing pullulan of the present invention has an advantageously significant feature that it has a relatively high rupture strength of piercing when formed into a film, though the particulate composition is so called "General-Purpose Industrial Product" produced without employing any purification step for pullulan, such as solvent sedimentation or the like.
  • the present invention solves the above object by providing a process for producing a particulate composition containing pullulan characterized in that it contains the steps of culturing a mutant of a microorganism of the species Aureobasidium pullulans in a culture medium containing glucose and maltose as carbon sources; removing the cells from the resulting culture; and decoloring, desalting, concentrating, and pulverizing the resultant, without employing a step of removing concomitant saccharides.
  • anymutants of amicroorganismof the species Aureobasidium pullulans can be used in the process of the present invention as long as they can produce the particulate composition containing pullulan of the present invention without employing any removing step for removing concomitant saccharides, and they should not be limited to specific ones.
  • Preferred examples of such mutants include the later described mutant of Aureobasidium pullulans S-2 strain (deposited in International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology under the accession number of FERM BP-11261). Based on the disclosure of the present specification, these mutants can be obtained by using the above S-2 strain as a parent strain and applying thereto conventional mutation method and the later described screening method.
  • S-2 strain is randomly mutated by conventional mutation method, followed by screening the resulting mutants with an index of the content of concomitant saccharides other than pullulan contained in the resulting cultures, and selecting the ones with significantly lower concomitant saccharide contents than that of S-2 strain as their parent strain.
  • mutant MA446 strain deposited in International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology under the accession number of FERM BP-11250.
  • the particulate composition containing pullulan of the present invention which is an inexpensive product produced without employing any complicated purification step of solvent precipitation or the like.
  • the particulate composition has the merit that it is more extensively used when formed into, for example, a pullulan film.
  • the process for producing the particulate composition containing pullulan of the present invention there is obtained the merit that the above-identified particulate composition containing pullulan with the above-mentioned advantageous merit can be obtained easily and cheaply without employing any complicated purification step of solvent precipitation or the like.
  • the present invention solves the above object by providing a shaped product containing pullulan produced by using at least partly the particulate composition containing pullulan of the present invention as a material.
  • the particulate composition containing pullulan of the present invention is so called "General-Purpose Industrial Product" produced without employing any purification step for pullulan, such as solvent precipitation, it has an advantageous feature that it imparts a relatively high rupture strength of piercing when formed into a film.
  • the particulate composition containing pullulan of the present invention has the following features so that it can be used as a pharmaceutical additive in pharmaceuticals, quasi-drugs, cosmetics, etc., which require a constant disposition of effective ingredients; the concomitant saccharide content is extremely as low as 3% by weight or lower, the saccharide composition is almost stably retained independently of its production lot because it has a relatively high purity of pullulan, and the rupture strength and the physical properties such as solubility and adhesive power less vary and less differ.
  • the particulate composition can be formed into films, as well as, for example, sheets, tablets, granules, and fibers, or coating agents, sugar coating agents, diluents/fillers/adjuvants, adhesive agents, and solid preparations such as a solid preparation to be reconstituted upon use directed for use in liquids. Accordingly, the particulate composition can provide shaped products which have constant and persistent properties required for pharmaceuticals, quasi-drugs, cosmetics, etc.
  • the particulate composition containing pullulan of the present invention when used in pharmaceuticals, can provide products that each have lesser dispersion in terms of the aspects of physical properties such as dissolution rate, integration rate, and rupture strength; and it has a lesser fear of affecting the disposition of effective ingredients due to dissolution or disintegration thereof.
  • the particulate composition can be used as a pharmaceutical additive.
  • the particulate composition containing pullulan of the present invention can be also used as a material for solid medicines in the form of a solid preparation to be reconstituted upon use directed for use in injections, etc., by applying thereunto a conventional method in general to make pyrogen free.
  • the particulate composition containing pullulan of the present invention is characterized in that it is produced by culturing a mutant of a microorganism of the species Aureobasidium pullulans in a culture medium containing glucose and maltose as carbon sources without employing a step of removing concomitant saccharides, it consists of a pullulan fraction and a concomitant fraction which are respectively insoluble and soluble in 75% by volume of methanol in water; it has a ratio of 3% by weight or lower of concomitant saccharides against the content of total saccharides contained in the whole particulate composition; and it contains mannitol.
  • the term "75% by volume of methanol in water” means a mixture that contains water and methanol in a volume ratio of 1:3.
  • any mutants can be used in the present invention.
  • parent strains usable in the present invention include Pullularia sp. S-2 strain, a pullulan producing microorganism, disclosed by Seinosuke UEDA in " Kogyo-Kagaku-Zasshi” (The Journal of Chemical Industry), Vol. 67, pp. 757-760, 1964 .
  • the microorganisms of the genus Pullularia are classified into those of the genus Aureobasidium and there has only been known one species, Aureobasidium pullulans. Since S-2 strain produces pullulan and, as described later, it has the cultural characteristics that well coincide with those of Aureobasidium pullulans , it has been identified as a microorganism of the species Aureobasidium pullulans and deposited on June 28, 2010 in International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Center 6, 1-1, Higashi 1-chome Tsukuba-shi, Ibaraki-ken, Japan, under the accession number of FERM BP-11261.
  • the above-identified S-2 strain is treated with mutation treatment conducted conventionally in the art such as exposure to ultraviolet rays and agent treatments with mutation-inducible agents, for example, as disclosed in Sumita SUGIYAMA et al., "Shin-Pan-Biseibutsu-Kagaku-Jikken-Ho", pp.
  • Examples of the desirable mutants usable in the present invention are those which the content of concomitant saccharides is lower than that contained in the resulting culture medium when they are cultured in a selection culture medium containing glucose and maltose as carbon sources, and more preferably, those which the content of concomitant saccharides is not higher than 50% of that of the culture of S-2 strain as a parent strain.
  • the particulate composition containing pullulan of the present invention should never be restricted to those which are produced from the culture of a specific mutant
  • concrete examples of preferable mutants that can produce the particulate composition containing pullulan of the present invention include Aureobasidium pullulans MA446 strain obtained by mutating the above Aureobasidium pullulans S-2 strain.
  • the mutant MA446 strain produces pullulan and has cultural characteristics well coincided with those of Aureobasidium pullulans , therefore, it was identified as a microorganisms belonging to Aureobasidium pullulans and has been deposited on April 30, 2010 in International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Center 6, 1-1, Higashi 1-chome Tsukuba-shi, Ibaraki-ken, Japan, under the accession number of FERM BP-11250.
  • S-2 strain as a parent strain and mutant MA446 strain have the following cultural characteristics:
  • Any culture media can be used in culturing mutants as long as they can produce the particulate composition containing pullulan of the present invention basically without employing a step of removing concomitant saccharides from the resulting cultures which contain lesser amounts of concomitant saccharides, and further those which appropriately contain carbon sources, nitrogen sources, organic nutrition sources, inorganic substances, etc., similarly as those which are used in culturing a microorganism of the species Aureobasidium pullulans for producing pullulan.
  • Examples of the carbon sources include saccharide mixtures containing glucose and maltose, preferably, saccharides which contain glucose and maltose having a dextrose equivalent (abbreviated as "DE”, hereinafter) of 50 to 90, and more preferably, saccharides which contain glucose and maltose in respective amounts of at least 10% by weight, d.s.b.
  • DE dextrose equivalent
  • the content of concomitant saccharides does not become so lowered, resulting in a difficulty of producing the particulate composition containing pullulan of the present invention.
  • the yield of pullulan decreases to lower the content of pullulan in the resulting particulate composition containing pullulan and to relatively increase the content of concomitant saccharides.
  • the concentration of carbon sources in culture media is desirable to be 5 to 20 w/v %.
  • nitrogen sources include one or more ingredients selected from inorganic nitrogen sources such as ammonium salts and nitrates; and organic nitrogen sources such as glutamates, peptones, yeast extracts, and corn steep liquor.
  • inorganic nitrogen sources such as ammonium salts and nitrates
  • organic nitrogen sources such as glutamates, peptones, yeast extracts, and corn steep liquor.
  • phosphates, magnesium salts, and iron salts can be appropriately used.
  • the conditions of culturing microorganisms are desirably conducted aerobically while stirring or shaking under aeration conditions. As preferable culture temperatures, 27°C is suitable for the growth of Aureobasidium pullulans. As culturing time, it is preferably be conducted up to almost reach the maximum level of pullulan yield after consumption of carbon sources and to reach a roughly constant level of concomitant saccharides after decrease thereof.
  • continuous culture can be employed by adding appropriate pH controlling agents to the culture media or controlling the dilution rate of the culture liquids by extracting samples therefrom intervally or continuously and by appropriately supplying or supplementing thereunto a fresh preparation of a nutrient culture media.
  • Culture supernatants containing pullulan can be obtained from the resulting cultures by removing cells from the cultures with conventionally used appropriate methods such as centrifugation and filtration.
  • the particulate composition containing pullulan of the present invention can be obtained in conventional manner by decoloring, desalting, concentrating, drying, and pulverizing the culture supernatants.
  • Cation-exchange resins and anion-exchange resins are usually used in desalting treatment.
  • cation-exchange resins include commercialized cation-exchange resins such as "DIAION PK218" (Mitsubishi Chemical Co., Ltd., Tokyo, Japan), "DIAION SK-1B” (Mitsubishi Chemical Co., Ltd., Tokyo, Japan)
  • anion-exchange resins include commercialized anion-exchange resins such as "DIAION WA30” (Mitsubishi Chemical Co., Ltd., Tokyo, Japan), and "AMBERLITE IRA411” (Japan Organo Co., Ltd., Tokyo, Japan).
  • Drying and pulverization of the culture supernatants can be conducted based on conventional methods .
  • the resulting blocks can be pulverized into particulate compositions or the culture supernatants can be simultaneously dried and pulverized by a spray-drying method.
  • the particulate composition containing pullulan of the present invention thus obtained contains 3% by weight or lower of concomitant saccharides against the content of total saccharides contained in the whole particulate composition, in other words, it stably contains pullulan in an amount of 97% by weight or higher, and more preferably, the composition contains 1% by weight or lower of concomitant saccharides against the content of total saccharides contained in the whole particulate composition, in other words, it stably contains pullulan in an amount of 99% by weight or more and contains mannitol.
  • the concomitant saccharides in the concomitant saccharide fraction contain saccharides with a glucose polymerization degree of 3 to 90 in the particulate composition containing pullulan of the present invention.
  • saccharides with a glucose polymerization degree of 3 to 90 concretely means, as described in the later discussed Experiment 4, ingredients that are detected in the range of those with a molecular weight corresponding to a glucose polymerization degree of 3 to 90, when a concomitant saccharide fraction dissolvable in 75% by volume of methanol in water of the particulate composition is subjected to gel permeation chromatography (abbreviated as "GPC", hereinafter); namely, those which are detected in the range of molecular weight of 500 to 15, 000 daltons in view of the fact that the average molecular weight determined on GPC has a constant error.
  • GPC gel permeation chromatography
  • the content of saccharides with a glucose polymerization degree of 3 to 90 is preferably 2% by weight or lower of the content of total saccharides contained in the whole particulate composition.
  • the content of saccharides with a glucose polymerization degree of 3 to 90 exceeds 2% by weight, it is not desirable because a prescribed rupture strength of piercing may not be obtained when formed into a film.
  • the above saccharides with a glucose polymerization degree of 3 to 90 is deemed to be those which have basically a pullulan-like structure wherein maltotriose molecules are coupled in an ⁇ -1,6 linkage fashion due to the fact that they form both maltotriose as a main ingredient and maltotetraose in a relatively small amount when subjected to the action of pullulanase.
  • the saccharides are those which have a relatively low molecular weight and dissolve in 75% by volume of methanol in water, though they have a pullulan-like structure.
  • the culture of a microorganism of the species Aureobasidium pullulans usually contains mannitol as a metabolite thereof.
  • Mannitol is dissolvable in 75% by volume of methanol in water and it is contained in the particulate composition containing pullulan of the present invention, unless the concomitant saccharides are removed from the culture by solvent precipitation or the like.
  • the fact that mannitol is present in the particulate composition containing pullulan means that the particulate composition is the one produced without employing a step of removing concomitant saccharides by solvent precipitation or the like.
  • mannitol is not detected by the anthrone-sulfuric acid method; however, the mannitol in the particulate composition can be determined on the later described high-performance liquid chromatography (HPLC) analysis used in Experiment4.
  • HPLC high-performance liquid chromatography
  • the content of mannitol in the particulate composition containing pullulan of the present invention is usually 0.04% by weight or higher, d.s.b., and it never exceeds 2% by weight.
  • the molecular weight of pullulan as the major ingredient of the particulate composition containing pullulan of the present invention should not specifically be restricted to specific ones as long as such pullulan imparts a desired rupture strength of piercing when formed into a film, it is desirable to have a weight-average molecular weight, determined on GPC (designated as "Mw", hereinafter), of about 50,000 to 1,000,000 daltons, more preferably, about 50,000 to 500,000 daltons.
  • Mw weight-average molecular weight
  • the ratio of Mw against the number average molecular weight of pullulan is usually about 10 to 70 (Mw/Mn) .
  • the ratio of Mw/Mn should not specifically be restricted to specific ones as long as the desired rupture strength of piercing is obtained, when formed into a film; however, preferable ratios are 100 or lower, more preferably, those in the range of about 10 to about 70.
  • the ratio of Mw/Mn exceeds 100, namely when the particulate composition has a wide range of molecular weight distribution of pullulan, the membrane formability of the particulate composition is poor and, when formed into a film, any film with a stable, high rupture strength of piercing could not easily be obtained.
  • the particulate composition containing pullulan of the present invention has white appearance and satisfactory free-flowing-ability, and exhibits a preferable solubility in water similarly as in conventional particulate composition containing pullulan.
  • the particulate composition containing pullulan of the present invention can be used after mixing with one or more ingredients, used in the fields of food products, pharmaceuticals, quasi-drugs, and cosmetics in general, selected from the group consisting of other materials, for example, polysaccharides other than pullulan, bulking agents, excipients/adjuvants, fillers, viscosity-imparting agents, surfactants, foaming agents, antifoam agents, pH-controlling agents, stabilizers, flame retardants, mold release agents, antiseptics, colors, flavors, nutrients, preferences including tobaccos, taste-imparting agents, medicines, and physiologically active substances.
  • the film When formed into a film as a concrete example of a shaped product made by the particulate composition containing pullulan of the present invention, the film has a satisfactory rupture strength of piercing, has a satisfactory solubility similarly as in conventional particulate compositions containing pullulan, and has an advantageous durability in use.
  • the particulate composition containing pullulan of the present invention can be used as a material for films, sheets, and coatings used in the fields of food products, pharmaceuticals, cosmetics, etc.
  • surfactants containing fatty acid esters of saccharides such as sugar fatty acid ester as a release agent.
  • the particulate composition containing pullulan of the present invention has both a satisfactory solubility in water and a satisfactory high strength when formed into films or the like; has a high purity of pullulan in that the content of concomitant saccharides to the total saccharides is 3% by weight or lower and the pullulan content is 97% by weight or higher; has a roughly constant saccharide composition, and has a stably constant range of molecular weight distribution of pullulan per se. Therefore, when used in shaped products, the particulate composition is expected to impart a remarkably-constant fixed strength, dissolution rate, and disintegration rate to the shaped products.
  • the particulate composition containing pullulan of the present invention can be used in food products and also used as a pharmaceutical additive in pharmaceuticals, quasi-drugs, cosmetics, etc., where the disposition of effective ingredients thereof is required to be remarkably-consistent.
  • the particulate composition can be used in films, as well as shaped products such as fibers for sheets, gauzes, surgical strings, etc.; it can be used as excipients/adjuvants/fillers, adhesives or coating agents in preparing tablets and granules.
  • the particulate composition can be prepared into solid medicines in the form of a solid preparation to be reconstituted upon use for use in liquid preparations.
  • ingredients widely used in respective fields can be appropriately added to shaped products produced by using the particulate composition at least partly as a material thereof.
  • the above shaped products are cosmetics or their intermediates, they can be formed into, for example, packs, masks, bath salts, or cachou films.
  • Antiseptics such as paraoxybenzoic acid, benzalkonium chloride, and pentanediol
  • skin whitening agents such as albutin, ellagic acid, tetrahydrocurcuminoid, and vitamin P
  • anti-inframmatories such as glycyrrhizic acid and glycyrrhiza extract
  • cell activators such as lactoferrin, chondroitin sulfate, hyaluronic acid, KANKOSO-101, and KANKOSO-301
  • humectants such as elastin, keratin, urea, and ceramide
  • oil-based medicines such as squalane, petrolatum, and tri-2-ethyl hexanoic acid cetyl
  • water-soluble high molecules such as carrageenan, carboxymethyl cellulose, locust bean gum, and carboxy vinyl polymer
  • the above shaped products are pharmaceuticals, quasi-drugs, or intermediates thereof, they can be formulated into granules, tablets, sugar-coated tablets, etc.
  • Immunosuppresants such as azathioprine, cyclosporine, cyclophosphamide, methotrexate, tacrolimus hydrate, and busulfan; anticancer agents such as capecitabine, rituximab, trastuzumab, bevacizumab, docetaxel, imatinib mesylate, 5-fluorouracil, anastrozole, taxol, tamoxifen, docetaxel, and hydroxycarbamide; anti-viral agents such as abacavir sulfate, zalcitabine, didanosine, famciclovir, and ribavirin; antibiotics such as amoxicillin, talampicillin
  • the above-identified shapedproduct such as cosmetics, pharmaceuticals, quasi-drugs, and intermediates thereof can be, if necessary, arbitrarily admixed with other high molecular substances, appropriate plasticizers, or fillers/excipients/adjuvants, etc., which are generally used in the fields of cosmetics, pharmaceuticals, and quasi-drugs, along with the particulate composition containing pullulan of the present invention.
  • the particulate composition containing pullulan of the present invention can be used as an adhesive.
  • high molecular substances examples include polysaccharides or derivatives thereof such as carrageenan, xanthan gum, carboxymethyl cellulose, cellulose, hemicelluloses, gum arabic, guar gum, carrageenan, pectin, agarose, dextrin, amylose, and starch containing processed starch; and proteins such as gelatin and casein.
  • polysaccharides or derivatives thereof such as carrageenan, xanthan gum, carboxymethyl cellulose, cellulose, hemicelluloses, gum arabic, guar gum, carrageenan, pectin, agarose, dextrin, amylose, and starch containing processed starch
  • proteins such as gelatin and casein.
  • plasticizers examples include saccharides such as maltitol, mannitol, maltitol, sucrose, maltose, lactose, ⁇ , ⁇ -trehalose, ⁇ , ⁇ -trehalose, gum arabic, corn starch, and crystalline cellulose; and inorganic substances such as aluminum hydroxide, calcium hydroxide, magnesium hydroxide, barium hydroxide, calcium sulfate, calcium sulfite, calcium carbonate, silica, calcium silicate, basic magnesium carbonate, kaolin, and talc.
  • ⁇ , ⁇ -trehalose can be advantageously used because it inhibits the deterioration of effective ingredients due to their oxidation decomposition and has functions of stably retaining the activity of the effective ingredients.
  • Conidiophores of Aureobasidium pullulans S-2 strain were irradiated with ultraviolet rays to induce mutation and inoculated to potato-dextrose agar plate, followed by selecting as a mutant a colony that had grown at 27°C.
  • the separated colony and S-2 strain as a parent strain were respectively inoculated to a liquid selection medium (pH 7.0) containing 10.0 w/v %, d.s.b., of a glucose syrup resolved by acid (DE 48, containing, on a dry solidbasis, about 27% glucose and about 17% maltose, commercialized by Hayashibara Shoji, Co., Ltd., Okayama, Japan), 0.2 w/v % dibasic potassium phosphate, 0.2 w/v % peptone, 0.2 w/v % sodium chloride, 0.04 w/v % magnesium sulfate heptahydrate, and 0.001 w/v % ferrous sulfate heptahydrate, and cultured under shaking conditions at 27°C for three days.
  • a liquid selection medium pH 7.0
  • a glucose syrup resolved by acid DE 48, containing, on a dry solidbasis, about 27% glucose and about 17% maltose, commercialized by
  • the culture supernatants which had been obtained by centrifuging each resulting culture to remove cells, were diluted with water by 10 times.
  • 3-times volume of methanol a special grade reagent, commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan
  • methanol a special grade reagent, commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan
  • the test solution corresponds to a dilute that had been prepared by diluting each supernatant by 400-times.
  • a 0.01 w/v % aqueous glucose solution was provided as a standard solution and water was used as a blank control, and each test solution was subjected to color reaction based on the anthrone-sulfuric acid method in accordance with the description of " Japanese Standard of Food Additives", 8th edition, pp. 572-573, 2007, published by Japan Food Additives Association , followed by measuring the absorbance of the resultant mixture at a wavelength of 620 nm by using a spectrophotometer and determining the concentration (%) of concomitant saccharides of each supernatant based on the following Equation 1.
  • the concentration of total saccharides in culture medium prior to cell inoculation was subjected to the anthrone-sulfuric acid method for determining the concentration (%) of carbon sources, followed by determining, as the yield (%) of concomitant saccharides and the yield (%) of pullulan against saccharides, the concentration (%) of concomitant saccharides and the concentration (%) of pullulan against the concentration (%) of carbon sources in the culture medium, determined based on by the following Equations 4 and 5, respectively.
  • these mutants showed almost the same level of the yield of pullulan against saccharides as that of S-2 strain as a parent strain, and the level of each of the strains is as low as 3.0% or lower that is lower than half of that (the concentration of concomitant saccharides: 6.6%) of S-2 strain as a parent strain, while the ratio of "the yield (%) of concomitant saccharides” against “the yield (%) of pullulan against saccharides” for each strain is respectively 0.05 or lower that is less than half of that (0.101) of S-2 strain.
  • These mutants shown in Table 1 have roughly the same level of the yield of pullulan against saccharides as those of their respective parent strains and have a lower yield of concomitant saccharides.
  • the starch syrup prepared by acid saccharification contains, on a dry solid basis, about 25% by weight of glucose and about 16% by weight of maltose.
  • MA446 Strain which had been cultured on a potato-dextrose agar slant at 27°C, was subjected to culture under rotatory shaking conditions in a liquid culture medium (pH 7.0) containing 10.0 w/v % sucrose, 0.2 w/v % dibasic potassium phosphate, 0.2 w/v % peptone, 0.2 w/v % sodium chloride, 0.04 w/v % magnesium sulfate heptahydrate, and 0.001 w/v % ferrous sulfate heptahydrate at 27°C at 230 rpm for 48 hours for use as a seed culture.
  • a liquid culture medium pH 7.0
  • culture media 1 to 5 containing 10.0 w/v % of any one of the above carbon sources, 0.2 w/v % dibasic potassium phosphate, 0.2 w/v % peptone, 0.2 w/v % sodium chloride, 0.04 w/v % magnesium sulfate heptahydrate, and 0.001 w/v % ferrous sulfate heptahydrate were prepared in a volume of 20 L per one 30 L-culture fermentor, adjusted to pH 7.0 and sterilized at 121°C for 15 min. The resulting cultures were respectively placed in a fermentor in a volume of 1,000 ml, and cultured at 27°C for three days at an aeration rate of 5.0 L/min and a rotation speed of 400 rpm.
  • Each resulting culture was centrifuged at 8,000 rpm to remove cells to obtain a supernatant, followed by decoloring treatment with filtration using an activated charcoal and desalting treatment with a cation exchange resin, "DIAION SK1B (H + -form)", Mitsubishi Chemical Co., Ltd., Tokyo, Japan, and an anion-exchange resin, "DIAION WA30 (OH - -form)", Mitsubishi Chemical Co., Ltd., Tokyo, Japan. After filtration for finishing with an activated charcoal, the resulting solutions were concentrated in vacuo up to give a concentration of about 25% by weight, d.s.b. The resulting each concentrate was spray dried to obtain a particulate composition containing pullulan.
  • S-2 Strain as a parent strain was cultured in the culture medium 1 similarly as in the above, and treated similarly as above to obtain a particulate composition containing pullulan for use as a control particulate composition containing pullulan.
  • S-2 strain was similarly cultured as above in the culture medium 1, and the resulting particulate composition containing pullulan has substantially the same pullulan content and the concomitant saccharide content as those of the above-identified "Food Additive Pullulan", and the above control as the particulate composition containing pullulan corresponds thereunto.
  • each material sample solution was diluted by 4.1 times.
  • a saturated potassium solution To one milliliter of each material sample solution was added 0.1 ml of a saturated potassium solution, the resulting mixture was admixed well with three milliliters of methanol, and centrifuged at 4°C and 15,700 g x 10 min to obtain a supernatant for use as a test solution (where each material sample solution was diluted by 4.1 times) .
  • concomitant saccharide and the pullulan content were determined based on the ratio of the absorbance of each sample solution and each standard material solution, where the ratio was not determined based on D-glucose equivalence by using D-glucose as a standard substance, however, the concomitant saccharide content and the pullulan content were coincided with those determined based on D-glucose equivalence, and each of their contents are expressed with "% by weight”.
  • MA446 strain was cultured in the culture medium 5 with DE 100 that consisted of glucose as carbon source, it showed the pullulan content of 95.5% by weight and the concomitant saccharide content of 4.5% by weight, wherein the pullulan content was increased and the concomitant saccharide was decreased compared to those of S-2 strain as a parent strain that was cultured in the culture medium 1. These differences were not so distinct as in the case of the culture media 2 to 4.
  • the particulate composition containing pullulan and the one as a control obtained in Experiment 2-2 were respectively dissolved in deionized water into a 20 w/v % aqueous solution, allowed to stand at 62.5°C and deaerated. An adequate amount of each of the resulting deaerated aqueous solutions was dropped and casted on a vinyl chloride plain plate, dried overnight at 25°C and a relative humidity of 30% to form a film with a thickness of about 100 ⁇ m. The formed film was detached from the plate and cut into a circle with a diameter of 19 mm for use as a test sample which was then conditioned for humidity at an ambient temperature and a relatively humidity of 22% for five days before subjecting it to a piercing test for rupture strength.
  • the piercing test for rupture strength was conducted with "Rheometer CR-500DX" (commercialized by Sun Scientific Co., Ltd., Tokyo, Japan), installed with an adaptor for piercing test with a cross-section area of 1 mm 2 in such a manner of perpendicularly pressing the adaptor on the center of the above film, fixed on the apparatus at a velocity of 50 mm/min to cause subsidial fracture, followed by determining the stress for rupture strength of piercing.
  • 10 sheets of each of the films were determined for rupture strength of piercing and the values were averaged.
  • any of the films, which had been formed with the particulate composition containing pullulan that had been produced by culturing MA446 strain in the culture media 2 to 4 showed a rupture strength of piercing of over 20 MPa.
  • the incidence of cracking as in the case of the cultures with the culture media 2 to 4 are all 0% that is extremely low level compared to the percentage of 50% of the culture of S-2 strain with the culture medium 1 as a control.
  • the fact that the incidence of cracking is low means that, even if rupture occurs, such rupture less extends there around and the film, which has 0% of the incidence of cracking and which is formed with the particulate composition produced with any of the cultures of MA446 strain with the culture media 2 to 4, has an advantageous feature in terms of strength.
  • any of the particulate compositions containing pullulan prepared from the cultures, obtained by culturing S-2 strain or IFO 6353 strain in the culture media 2 to 4 have a pullulan content of below 95% by weight and a concomitant saccharide content of about 6% by weight or higher, the levels of which are respectively distinctly lower and higher than that prepared from the culture of MA446 strain.
  • the film formed with the particulate composition containing pullulan obtained by culturing S-2 strain or IFO 6353 strain in the culture media 2 to 4 was ruptured by the stress below the level of 18 MPa on the piercing test for rupture strength.
  • the rupture strength of piercing significantly exceeds the level of over 21 MPa that attained by the film formed with the particulate composition containing pullulan obtained by culturing MA446 stain in the culture media 2 to 4.
  • the film formed with the particulate composition containing pullulan, obtained by culturing S-2 strain or IFO 6353 strain in any of the culture media 2 to 4 shows an incidence of cracking of 30% or higher, meaning that the rupture easily spreads there around when it once occurs.
  • the particulate composition containing pullulan which had been obtained in Experiments 2 to 4 and had the highest rupture strength of piercing, i.e., the one that had been prepared from the culture that had been obtained by culturing MA446 strain in the culture medium 3, were measured for molecular weight distribution on GPC.
  • concomitant saccharides soluble in 75% by volume of methanol in water were prepared therefrom and subjected to GPC analysis. The content of mannitol contained therein was assayed on HPLC.
  • test powder and the control powder were respectively prepared into a 2 w/v % aqueous solution and admixed with an equal amount of 20 mM phosphate buffer (pH 7.0), and the resulting mixture was membrane filtered for use in GPC analysis.
  • 20 mM phosphate buffer (pH 7.0) Upon GPC analysis, two columns packed with TSK-GEL ⁇ -M (7.8 mm ⁇ inner diameter x 300 mm length, Tosoh Corporation, Tokyo, Japan) cascaded in series, 10 mM phosphate buffer (pH 7.0) as an eluate, and a differential refractometer as a detector were used, and it was carried out at a temperature of 40°C and a flow rate of 0.3 ml/min.
  • FIGs. 1 and 2 are respectively the GPC elution patterns of the test powder and the control powder.
  • FIG. 5 shows the values of weight-average molecular weight and Mw/Mn.
  • the GPC elution pattern of the test powder consists of a single peak of pullulan that exhibits the top at a retention time of about 50min, and does not substantially contain the ingredients corresponding to the retention times of less than 40 min and over 60 min, meaning that the test powder is of which has a high pullulan purity. While, in the elution pattern of the control powder in FIG. 2 , a peak of pullulan that has the top at a retention time of about 50 min is observed, however, several peaks can be observed at the positions corresponding to the retention times of over 60 min, meaning that the control powder contains plenty of concomitant saccharides, other than pullulan, with a lower molecular weight than the pullulan.
  • concomitant saccharide fractions that are soluble in 75% by volume of methanol in water were prepared as follows: A test powder and a control powder were respectively prepared into a 10 w/v % aqueous solution which was then admixed with 3-times volume of methanol, and the resulting mixture was promptly well mixed, and centrifuged at 4°C and 15,700 g x 10 min. Each supernatant was dried by an evaporator, admixed with refined water to dissolve the contents, and dried.
  • FIGs. 3 and 4 are respectively the GPC elution patterns of the concomitant saccharide fractions of the test powder and the control powder.
  • the retention times ranging from about 60 min to about 70 min correspond to the molecular weights ranging from about 500 to about 15,000 and roughly correspond to saccharides having a pullulan-like structure, where maltotriose molecules are coupled in series in an ⁇ -1,6 linkage fashion, with a glucose polymerization degree of about 3 to about 90 (ranging from 1 (molecular weight of 504) to 30 (molecular weight of 14,598) by maltotriose unit).
  • the peak observed at a retention time of about 71 min corresponds to mannitol as a metabolite of MA446 strain and S-2 strain used in the formation of pullulan in producing the test powder and the control powder.
  • the concomitant saccharide fractions of the test powder and the control powder contain mannitol, meaning that both the test powder and the control powder are produced without employing a removing step for removing concomitant saccharides by solvent sedimentation.
  • mannitol is not assayed on the anthrone-sulfuric acid method and is not contained in concomitant saccharides, however, it is detected as a peak at a retention time of about 71 min on the GPC analysis of concomitant saccharide fraction.
  • the percentage of the area of a peak, corresponding to the saccharides with a glucose polymerization degree of 3 to 90, against the above-identified peak area is determined and shown in Table 5 as the content of saccharides with a glucose polymerization degree (DP) of 3 to 90 ("saccharide content (%) with a DP of 3 to 90 in concomitant saccharides”) measured on the anthrone sulfuric acid method.
  • DP glucose polymerization degree
  • saccharide content (%) with a DP of 3 to 90 in concomitant saccharides is multiplied by the concomitant saccharide content (%) against the total saccharide content contained in the whole powder that is alternatively determined on the anthrone-sulfuric acid method, and the resultant value is regarded as "saccharide content (%) with a DP of 3 to 90 inparticulate composition containing pullulan” and is shown in Table 5 in parallel.
  • the test powder and the control powder were analyzed on HPLC to determine the mannitol contents in the particulate compositions containing pullulan.
  • the HPLC analysis was conducted by using a column of MCI GEL CK08EC (8 mm ⁇ inner diameter x 300 mm length, Mitsubishi Chemical Co., Ltd., Tokyo, Japan), refined water as an eluant, and a differential refractometer as a detector, and the elution conditions were 75°C and an eluant flow-rate of 0.6 ml/min.
  • MCI GEL CK08EC 8 mm ⁇ inner diameter x 300 mm length, Mitsubishi Chemical Co., Ltd., Tokyo, Japan
  • refined water refined water
  • a differential refractometer as a detector
  • the elution conditions were 75°C and an eluant flow-rate of 0.6 ml/min.
  • the mannitol content was determined, on a dry solid basis, based on the peak area in the particulate composition containing
  • the test powder produced from the culture of MA446 strain showed no great difference in both the average-molecular weight and the molecular weight distribution compared to those of the control powder.
  • the content of saccharides with a glucose polymerization degree (DP) of 3 to 90 in the concomitant saccharides was 24.2% by weight (the content of saccharides with a DP of 3 to 90 in the particulate composition containing pullulan is 0.1% by weight), which was lower than 85.1% by weight for the control powder (the content of saccharides with a DP of 3 to 90 in the particulate composition containing pullulan is 6.5% by weight) .
  • DP glucose polymerization degree
  • the content of mannitol in particulate composition containing pullulan of the test powder and the control powder were respectively 0.35% by weight and 0.51% by weight.
  • the content of saccharides with a DP of 3 to 90 in concomitant saccharides is also low, compared to the control powder.
  • test powder and the control powder which had been used in Experiment 4, were mixed in a ratio shown in Table 6 to prepare test samples 1 to 10 with different contents of concomitant saccharides.
  • Table 6 shows the content of pullulan, the content of concomitant saccharides, and the content of saccharides with a glucose polymerization degree (DP) of 3 to 90.
  • DP glucose polymerization degree
  • the films which had been formed with the test samples 1 to 4 having a pullulan content of 97% by weight or higher and the concomitant saccharide content of 3% by weight or lower, showed a higher rupture strength of piercing compared to the film which had been formed with the test sample 10 consisting of the control powder, and did not cause rupture even when subjected to a stress of 20 MPa. While in the case of the test samples 5 to 9 with a pullulan content of less than 97% by weight and a saccharide concomitant content of over 3% by weight, the films formed therewith were ruptured when subjected to a stress of below 20 MPa.
  • the content of saccharides with a glucose polymerization degree of 3 to 90 is 2% by weight or lower.
  • the content of saccharides with a glucose polymerization degree of 3 to 90 should preferably be a level of 2% by weight or lower.
  • Diffraction patterns obtained from the above analysis are shown in FIGs. 5 and 6 .
  • FIGs. 5 and 6 in any one of diffraction patterns, scattering curves smoothly attenuate without creating any kinks.
  • a particulate composition containing pullulan, which had been obtained by culturing MA446 strain in the culture medium 3 according to the method in Experiment 2, and the particulate composition containing pullulan used as the control in Experiment 4 were respectively prepared into a 10 w/v % aqueous solution which was then admixed with 3-times volume of methanol, followed by promptly mixing the mixture and centrifuging the resulting mixture at 4°C and 15,700 g x 10 min. The resulting precipitate was redissolved in water and re-treated with the above procedure.
  • the precipitate thus obtained was dried, dissolved in 20 mM acetate buffer (pH 5.0) to make into a 1 w/v % solution which was then admixed with "AMANO 3", a pullulanase specimen commercialized by Amano Enzyme Inc., Ltd., Aichi, Japan, in an amount of 5 units/g particulate composition, followed by an enzymatic reaction at 53°C for 48 hours.
  • the resulting reaction solution was desalted, appropriately diluted, and analyzed on HPLC to determine the yields of maltotriose and maltotetraose.
  • the HPLC analysis was conducted by using a column of MCI GEL CK04SS (8 mm ⁇ inner diameter x 300 mm length, Mitsubishi Chemical Co., Ltd., Tokyo, Japan), refined water as an eluant, and a differential refractometer as a detector, and the elution conditions were 75°C and an eluant flow-rate of 0.6 ml/min. Based on the peak areas of maltotriose and maltotetraose obtained in the chromatogram, each content of the saccharides was determined and, based on the data, their molar ratio was determined. The results are in Table 8. Table 8 Particulate composition containing pullulan Test powder Control powder Maltotriose (G3)(%) 97.8 97.2 Maltotetraose (G4)(%) 1.8 2.3 G4/G3(molar %) 1.4 1.8
  • the maltotetraose structure is present in a molar ratio of 1% to 3% against the maltotriose structure.
  • the particulate composition containing pullulan obtained from the culture of MA446 strain makes a little difference in terms of the ratio of the maltotetraose structure against the maltotriose structure, compared to the particulate composition containing pullulan as the control powder.
  • the film of the particulate composition containing pullulan of the present invention has a higher rupture strength of piercing and a lesser incidence of cracking is not due to the structural difference but due to the concomitant saccharide content.
  • a culture medium (pH 7.0), which had been finally volumed up with water and contained as carbon sources 50 parts by weight of glucose, 50 parts by weight of maltose, and 50 parts by weight of a starch syrup ("MALT-RUP ® ", an enzymatically saccharified starch syrup, a solid content of 80%, a DE of about 47, commercialized by Hayashibara Shoji, Co., Ltd., Okayama, Japan) (a DE of about 67 as carbon sources), and two parts by weight of dipotassium hydrogen phosphate, two parts by weight of peptone, two parts by weight of sodium chloride, 0.4 part by weight of magnesium sulfate heptahydrate, and 0.01 part by weight of ferrous sulfate heptahydrate, was inoculated a seed culture of Aureobasidium pullulans MA446 strain, which had been cultured in a fresh preparation of the same culture medium as in the above at 27°C for 48 hours, followed by cul
  • the cells were removed from the resulting culture by centrifugation, and the supernatant was decolored/ filtered with an activated charcoal, purified by desalting with an ion-exchange resin, concentrated, dried, and pulverized to obtain a particulate composition containing pullulan as a white powder having a satisfactory free-flowing ability.
  • the particulate composition had a pullulan content of 99.2% by weight, a concomitant saccharide content of 0.8% by weight, and a content of saccharides with glucose polymerization degrees of 3 to 90 of 0.3% by weight. Also, the particulate composition contains 0.2% by weight of mannitol, d.s.b. Further, the average molecular weight of the particulate composition was 428,000 daltons and the ratio of Mw/Mn was 35.3. The particulate composition can be formed into a film with a satisfactory rupture strength.
  • the particulate composition has a lesser amount of concomitant saccharides, and the ratio of Mw/Mn as an index for broadness of molecular weight distribution is 35.3 as in the range of 10 to 70, indicating that it is expected to have a satisfactory stable properties in terms of physical properties such as the strength of shaped products, as well as its dissolution and disintegration rates.
  • the particulate composition can be used in foodproducts, as well as cosmetics, pharmaceuticals, quasi-drugs, etc.
  • the particulate composition can be preferably used as a pharmaceutical additive because it does not affect disposition of effective ingredients due to dispersion of solubility and disnintegrability.
  • the cells were removed from the resulting culture by centrifugation, and the supernatant was decolored/filtered with an activated charcoal, purified by desalting with an ion-exchange resin, concentrated, dried, and pulverized to obtain a particulate composition containing pullulan as a white powder having a satisfactory free-flowing ability.
  • the particulate composition had a pullulan content of 97.9% by weight, a concomitant saccharide content of 2.1% by weight, and a content of saccharides with glucose polymerization degrees of 3 to 90 of 0.7% by weight. Also, the particulate composition contains 0.6% by weight of mannitol, d.s.b. Further, the average molecular weight of the particulate composition was 401,000 daltons and the ratio of Mw/Mn was 41.1. The particulate composition can be formed into a film with a satisfactory rupture strength.
  • the particulate composition has a lesser amount of concomitant saccharides, and the ratio of Mw/Mn as an index for broadness of molecular weight distribution is 41.1 as in the range of 10 to 70, indicating that it is expected to have a satisfactory stable properties in terms of physical properties such as the strength of shaped products, as well as its dissolution and disintegration rates.
  • the particulate composition can be used in food products, as well as cosmetics, pharmaceuticals, quasi-drugs, etc.
  • the particulate composition can be preferably used as a pharmaceutical additive because it does not affect disposition of effective ingredients due to dispersion of solubility and disnintegrability.
  • the pullulan film has an improved tolerance against a rupture stress and a high strength, has a satisfactory solubility in water, has a stable dissolution rate with a lesser dispersion independently of its production lot, and therefore it can be advantageously used in food products, cosmetics, pharmaceuticals, etc.
  • the resulting mixture was dissolved by stirring at 90°C for three hours, casted on a 2 ⁇ 10 m stainless steel plate and dried at 60°C for four hours to obtain a film-shaped product with an about 200 ⁇ m thick, about 200 cm wide, 10 m length, about 8% moisture content, and about 2.2 g per 100 cm 2 .
  • the film-shaped product was cut into a size of 1 ⁇ 2 cm, and 20 sheets of which were packed in a portable container to obtain a mouth refreshing film.
  • the product is an edible film which has an appropriate strength and a prompt solubility in the mouth and contains glycosyl hesperidin and propolis extract, it is a cachou film that can be used for the purpose of maintaining/promoting the health in the mouth.
  • the product is an edible film which is produced with the particulate composition containing pullulan of the present invention as a material that has a roughly constant saccharide composition being less varied batch-by-batch, and therefore the dissolution rate in the mouth is roughly constant, and the rate of dissolving the effective ingredients such as glycosyl hesperidin is usually stable.
  • the material aqueous solution thus obtained was continuously casted on over a synthetic plastic film and dried by passing through a 50°C air stream to obtain a pullulan sheet with 100 ⁇ m thick.
  • the pullulan sheet is produced with the particulate composition containing pullulan of the present invention as a material that has a roughly constant saccharide composition being less varied batch-by-batch, and therefore it has an improved rupture strength, a satisfactory handleability, and an expected constant dissolution rate, independently of its production lot.
  • the product can be suitably used as a processing material for cosmetic pack that allows effective ingredients such as L-ascorbic acid 2-glucoside and ⁇ -glucosyl rutin to act on the skin at a stable, constant rate.
  • An aqueous solution was prepared by dissolving in 100 parts by weight of water one part by weight of a particulate composition containing pullulan obtained by the method in Example 1 and 0.2 part by weight of gumarabic. A fresh egg within 10 hours of postlaying was soaked in the aqueous solution for 30 sec, taken out from the solution, dried at 30°C for two hours to form a pullulan membrane on the egg shell.
  • the egg coated with the pullulan membrane was stored at ambient temperature (15 to 25°C), and the edible period was compared with an untreated egg as a control (with no pullulan-membrane), revealing that the edible period of the egg coated with the pullulan membrane was prolonged even by about 5 to about 10 times.
  • the pullulan membrane can be advantageously used to store eggs for use in materials for food industry, etc.
  • a 150 mg crude agent as a wicking agent was coated with a coating solution which contained 50 parts by weight of crystalline maltitol, 20 parts by weight of a 10 w/w % aqueous solution prepared with a particulate composition containing pullulan produced by the method in Example 2, 15 parts by weight of water, 25 parts by weight of talc, and three parts by weight of titanium oxide until it reached about 230 mg weight.
  • the resulting agent was further coated with another solution which contained 65 parts by weight of a fresh preparation of the same crystalline maltitol as used in the above, 10 parts by weight of a fresh preparation of the same 10 w/w % aqueous pullulan solution as used in the above, and 25 parts by weight of water, and successively coated with a liquid wax to obtain a glossy sugar coated tablet with a satisfactory appearance.
  • the product is a sugar coated tablet that is coated with a membrane containing pullulan, has an improved tolerance against rupture strength, has a lesser damage when transported or packed, and retains the quality of effective ingredients contained in the wicking agent for a relatively long period of time. Since the product is produced with the particulate composition containing pullulan of the present invention as a material that retains the saccharide composition in such a manner of being less varied batch-by-batch and roughly consistent, both the dissolution rate after administration and the binding with the wicking agent are roughly constant and the effective ingredients contained in the wicking agent are absorbed by the body at a stable, constant rate.
  • the product is suitably used as an orally administrable product for promoting the health because it is a tablet that induces no crack when tabletted, has an adequate strength, exhibits a satisfactory dissolvability in water to ease its swallowing, and contains ⁇ -glycosyl rutin and propolis extract, as well as lactosucrose and pullulan.
  • the adhesive force inherent to the particulate composition containing pullulan is roughly constant because the product is produced with the particulate composition containing pullulan of the present invention as a material that retains the saccharide composition in such a manner of being less varied batch-by-batch and roughly consistent.
  • the product is a tablet, which has a constant dissolution rate and disintegrability after administration and which the effective ingredients are absorbed by the body at a stable, constant rate.
  • the product does not induce any cracking when tabletted, has an adequate strength, and has a satisfactory water solubility, it is suitably used as a vitamin agent.
  • the adhesive force inherent to the particulate composition containing pullulan is roughly constant because the product is produced with the particulate composition containing pullulan of the present invention as a material that retains the saccharide composition in such a manner of being less varied batch-by-batch and roughly consistent.
  • a satisfactory tablet which has a usually-stable shape and strength, can be obtained.
  • the product is a tablet, which has a constant dissolution rate and disintegrability after administration and which the effective ingredients are absorbed by the body at a stable, constant rate.
  • ⁇ G-SWEET a product name of ⁇ -glucosyl stevioside commercialized by Toyo Sugar Refining
  • the product does not induce any cracking when tabletted, has an adequate strength, and has a satisfactory water solubility, it can be used as a sublingual-type cold medicine that is injectable while dissolving in the mouth.
  • the adhesive force inherent to the particulate composition containing pullulan is roughly constant because the product is produced with the particulate composition containing pullulan of the present invention as a material that retains the saccharide composition in such a manner of being less varied batch-by-batch and roughly consistent.
  • a satisfactory tablet which has a usually-stable shape and strength, can be obtained.
  • the product has a constant dissolution rate in the mouth, the dissolution rates of the effective ingredients such as ethenzamide and acetaminophen are stable and they can be allowed to act on the body at a higher efficiency.
  • LS-90P a product name of lactosucrose-containing-saccharides with a lactosucrose content of about 90% by weight, d.s.b., commercialized by Ensuiko Sugar Refining Co., Ltd., Tokyo, Japan
  • 0.1 part by weight of a particulate composition containing pullulan obtained by the method in Example 2 0.5 part by weight of " ⁇ G-HESPERIDIN”, a product name of glycosyl hesperidin, commercialzied by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, and 0.2 part by weight of " ⁇ G-SWEET”, a product name of a glycosyl hesperidin specimen commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, were mixed to homogeneity and subjected to a granulator to obtain a granular powdered preparation.
  • the product contains pullulan, it has a satisfactory unity as a granular, improved safeness and dissolvability, and satisfactory swallowability.
  • the product can be useful as a health food for maintaining/promoting the health because it contains glycosyl hesperidin.
  • the adhesive force inherent to the particulate composition containing pullulan is roughly constant because the product is produced with the particulate composition containing pullulan of the present invention as a material that retains the saccharide composition in such a manner of being less varied batch-by-batch and roughly consistent.
  • the product is a granule, which has a constant dissolution rate and which the effective ingredients such as glycosyl hesperidin are stably absorbed by the body.
  • a particulate composition containing pullulan obtained in Example 1 was dissolved in water to obtain a 40 w/w % aqueous solution, into which were then dissolved 1.5 w/w %, d.s.b., of alginic acid and 0.02 w/w %, d.s.b., of locust bean gum, and the resulting solution was adjusted to 60°C, followed by pressing the solution into the atmospheric air through a cylindrical nozzle, 0.3 mm diameter, 1 mm length, at a pressure of 3 kg/cm 2 to form a strand and spooling it by a roller while drying to evaporate moisture.
  • the fiber thus obtained has a size of about 25 ⁇ m, and it can be twisted, knitted, and weaved. Also, the fiber has an adequate strength and hydrophilicity and it is characteristically, basically harmless and non stimulant to the skin because it is made from pullulan, it can be, for example, suitable for surgical strings, gauzes, etc.
  • the adhesive force and dissolvability inherent to the particulate composition containing pullulan is roughly constant because the product is produced with the particulate composition containing pullulan of the present invention as a material that retains the saccharide composition in such a manner of being less varied batch-by-batch and roughly consistent. Accordingly, the product is an advantageous fiber that can form fibrious shaped products that have a constant quality such as strength and dissolvability under a prescribed spinning conditions.
  • the product has a relatively long shelf life even when stored at ambient temperature.
  • the product promptly dissolves in water and it does not affect the disposition of interferon- ⁇ as the effective ingredient and retains it constantly because the product contains the particulate composition containing pullulan of the present invention wherein the saccharide composition less varies batch-by-batch and is kept roughly consistently.
  • the product can be used as an injection, reagent for test, etc.
  • a particulate composition containing pullulan with an improved features can be produced from a culture obtained by culturing a mutant of Aureobasidium pullulans without employing a complicated purification step such as solvent precipitation.
  • the particulate composition can be produced without a complicated purification step, has an extremely low level of concomitant saccharides in the total saccharides, has a relatively high purity of pullulan, has a stable composition, and has a molecular weight distribution of pullulan within a prescribed constant range.
  • the shaped bodies such as films formed with the particulate composition containing pullulan of the present invention has a satisfactory, constantly stable strength, dissolvability, and disintegration independently of their production lots, thus the particulate composition will greatly contribute to the expansion of the use of pullulan in the fields of food products, as well as pharmaceuticals, quasi-drugs, and cosmetics, as a relatively low-price, high-quality particulate composition containing pullulan.

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CN105467060B (zh) * 2015-11-30 2019-01-11 山东省药学科学院 一种普鲁兰多糖含量的快速检测方法
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JP2016145248A (ja) 2016-08-12
EP2583982A1 (en) 2013-04-24
US20190085367A1 (en) 2019-03-21
CN103140503A (zh) 2013-06-05
KR20130114077A (ko) 2013-10-16
JP5669838B2 (ja) 2015-02-18
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